Bacteria have played a crucial role in the development of the biotechnology. Since the development of recombinant DNA technology, genetically engineered bacteria have been employed for the production of various recombinant proteins. Also, recombinant bacteria have been developed and used for a wide range of industrial applications such as biodegradable plastics production, heavy metal removal, sulfur removal, waste treatment, and food processing.
Recently, new advances in molecular biology and secretory expression of proteins made it possible to express foreign proteins at the outer surface of microorganisms by the technology called cell surface display.
Since the first development of surface-expression system by George P. Smith in the mid 1980 by expressing peptides or small proteins fused with pIII of the filamentous phage (see: Smith, G. P., Science, 228:1315-1317, 1985), various mechanisms of protein secretion in microorganisms have been extensively studied to develop new and better cell surface display systems by which proteins of interest can be expressed on the surface of the microorganisms. Cell surface display is a relatively new technology expressing proteins or peptides on the surface of the cell in a stable manner using the surface protein of bacteria, yeast, or even mammalian cells as a surface anchoring motif.
Before the cell surface display system was developed, phage system was used to express foreign protein on the surface of the phage, because the structure of the phage coat is simpler than that of bacteria. However, the size of foreign protein to be expressed on the surface of phage was limited. Therefore, the application of the phage surface display system has been limited. This is why new focus has been given to bacterial cell surface display system.
Gram-negative bacteria possess unique as well as complex cell envelope structure which consists of inner cellular membrane, periplasm, and outer cellular membrane. Therefore, surface anchoring motif is needed to efficiently transport foreign protein to the surface of the bacteria. For the expression of foreign proteins or peptides using the surface protein of the bacteria, appropriate bacterial surface protein has to be fused to the foreign protein of interest at the genetic level, and the expressed fusion protein has to be transported through the inner cellular membrane and outer membrane to the surface and be maintained on the surface of the bacteria.
Successful candidates for surface anchoring motif should have the following characteristics: The surface protein to be used as an anchoring motif should have efficient secretion signal sequences for facilitating the penetration of the foreign protein through the inner membrane of the cell, targeting signal for anchoring foreign protein to the surface of the cell in a stable manner, and capacity to accommodate foreign proteins or peptides of various sizes. Furthermore, it would be beneficial if the fusion protein can be expressed in large amounts.
A variety of cell surface display systems have been developed to date, which may be classified into three groups according to their recombinant profiles: C-terminal fusion, N-terminal fusion, and sandwich fusion. If a native surface protein has a discrete localization signal within its N-terminal portion, a C-terminal fusion strategy may be considered to fuse the foreign peptides to the C-terminal of that function portion. The Lpp-OmpA motif developed in E. coli is a good example of C-terminal fusion system (see: Georgiou, G., et al., Protein Eng., 9:239-247, 1996). Similarly, N-terminal fusion systems have been developed using Staphylococcus aureus protein A (see: Gunneriusson, E., et al., J. Bacteriol., 178:1341-1346, 1996), Staphylococcus aureus fibronectin binding protein B (see: Strauss, A., et al., Mol. Microbiol., 21:491-500, 1996), and Streptococcus pyogenes fibrillar M protein (see: Pozzi, G., et al., Infect. Immun., 60:1902-1907, 1992), all of which contain C-terminal sorting signals to target foreign proteins to the cell wall. However, many surface proteins do not have such anchoring regions, and thus the whole structure is required for the assembly. Therefore, a sandwich-fusion system in which a foreign protein of interest is inserted into the surface protein has also been developed. Several examples employing this system include E. coli PhoE (see: Agterberg, M., et al., Gene, 88:37-45, 1990), FimH (see: Pallesen, L., et al., Microbiology, 141:2839-2848, 1995), and PapA (see: Steidler, L., et al., J. Bacteriol., 175:7639-7643, 1993). However, it is generally believed that the exposed loops of outer membrane proteins (OMPs) can only accept insertions of 60-70 amino acids or less (see: Georgiou, G., et al., Nature Biotechnol., 15:29-34, 1997; Stahl, S., et al., Trends Biotechnol., 15:185-192, 1997).
Under the current circumstances, there are strong reasons for exploring and developing an alternative cell surface display system to allow expression and display of foreign proteins consisted of more amino acid residues.
The cell surface display can be employed for a wide range of biotechnological and industrial applications such as:
(1) Live vaccine development--to expose heterologous epitopes on human commensal or attenuated pathogenic bacterial cells to elicit antigen-specific antibody responses (see: Nguyen, T. N., et al., Gene, 128:89-94, 1993); PA1 (2) Peptide libraries screening--to screen displayed peptide libraries by sequential binding and elution, or more efficiently, by fluorescence-activated cell sorting (see: Francisco, J. A., et al., Proc. Natl. Acad. Sci., USA., 90:10444-10448, 1993; Georgiou, G., WO9849286, 1998); PA1 (3) Antibody production--to express surface antigens to raise polyclonal antibodies in animal (see: Charbit, A., et al., Gene, 70:181-189, 1988); PA1 (4) Environmental bioadsorbents--to modify cell surface for the removal of harmful chemicals and heavymetals (see: Sousa, C., et al., J. Bacteriol., 180:2280-2284, 1998); PA1 (5) Whole cell catalysts--to immobilize enzymes on the outmost layer of cells to catalyze biochemical reactions directly (see: Richins, R. D., et al., Nature Biotechnol., 15:984-987, 1997); and, PA1 (6) Biosensor development--to anchor enzymes, receptors, or other signal-sensitive components on cell surface to develop novel biosensors for diagnostic, industrial or environmental purposes.